The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs

Article abstract of DOI:10.1016/j.bmcl.2013.11.067

Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and several reports have described potent Dxr inhibitors. M

Full text of DOI:10.1016/j.bmcl.2013.11.067

Bioorganic & Medicinal Chemistry Letters 24 (2014) 649–653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The effect of chain length and unsaturation on Mtb Dxr inhibition
and antitubercular killing activity of FR900098 analogs
Emily R. Jackson a, Géraldine San Jose a, Robert C. Brothers a, Emma K. Edelstein a, Zachary Sheldon a,
Amanda Haymond b, Chinchu Johny b, Helena I. Boshoff c, Robin D. Couch b, Cynthia S. Dowd a,
⇑
a Department of Chemistry, George Washington University, 725 21st Street NW, Corcoran 107, Washington, DC 20052, United States
b Department of Chemistry and Biochemistry and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, United States
c Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source
of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and sev-
eral reports have described potent Dxr inhibitors. Many of these compounds are structurally related to
natural products fosmidomycin and FR900098, each bearing retrohydroxamate and phosphonate groups.
We synthesized a series of compounds with two to five methylene units separating these groups to exam-
ine what linker length was optimal and tested for inhibition against Mtb Dxr. We synthesized ethyl and
pivaloyl esters of these compounds to increase lipophilicity and improve inhibition of Mtb growth. Our
results show that propyl or propenyl linker chains are optimal. Propenyl analog 22 has an IC50 of
Received 21 October 2013
Revised 20 November 2013
Accepted 25 November 2013
Available online 4 December 2013
Keywords:
Mycobacterium tuberculosis
Nonmevalonate pathway
Dxr
1.07
lM against Mtb Dxr. The pivaloyl ester of 22, compound 26, has an MIC of 9.4 lg/mL, representing
a significant improvement in antitubercular potency in this class of compounds.
Antibiotic
Ó 2013 Elsevier Ltd. All rights reserved.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb),
remains one of the world’s deadliest infectious diseases.1 Emer-
gence of multi-drug (MDR) and extensively-drug (XDR) resistant
strains, as well as co-infection with HIV, has made TB both difficult
and expensive to treat.2 New TB therapies are needed to shorten
treatment, be effective against all strains and metabolic states of
the organism, and work well with HIV drugs. Thus, there remains
a significant need for new and improved strategies against Mtb.
The nonmevalonate pathway (NMP) of isoprene biosynthesis
(Fig. 1) is essential for Mtb survival and, as it is not present in hu-
mans, is an attractive set of targets for novel drug development.3–5
The NMP synthesizes 5-carbon building blocks from pyruvate and
glyceraldehyde-3-phosphate. These building blocks are the starting
more effectively kill
a
range of bacterial strains, including
Mtb.10–12 Since that time, we and others have reported Dxr inhib-
itors belonging to several structural families,11,13–16 but very few of
these have displayed potent antitubercular activity. Many of these
inhibitors retain key structural features found in the parent com-
pounds 1 and 2: a retrohydroxamic acid, a phosphonate, and an
n-propyl carbon chain linking the nitrogen and phosphorus atoms.
In the 1980s, a series of Streptomyces-derived and inspired prod-
ucts exchanging the n-propyl chain for ethylene and propenyl
chains were described.17,18 Among these, the propenyl compound
was found to be comparable to the propyl analogs 1 and 2 and
showed potent antibacterial activity against Bacillus subtilis and
Escherichia coli.18 As this work came before the discovery of Dxr
as the cellular target of these inhibitors, the inhibitory activity of
these carbon chain-modified analogs against the purified enzyme
is largely unknown. To fill this gap and expand on the set of analogs
examined, we synthesized analogs of 1 and 2, varying the length of
the carbon linker from 2 to 5 methylene groups. We also prepared
the propenyl analog to examine the influence of unsaturation with-
in the propyl chain. As our interest is the development of antitu-
bercular agents working through Dxr inhibition, we evaluated
these analogs as inhibitors of Mtb Dxr. To study the effects of these
structural changes on antitubercular activity, the ethyl and
selected pivaloyl esters were prepared. The compounds synthe-
sized and evaluated are shown in Figure 2.
materials for many complex cellular metabolites. 1-Deoxy-
lose-5-phosphate reductoisomerase (Dxr), is the first committed
step in the NMP and is responsible for conversion of 1-deoxy-
D-xylu-
D
-
xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phos-
phate (MEP).6 Dxr catalyzes both a reduction and isomerization
using NADPH as a cofactor.
Natural products fosmidomycin (1) and FR900098 (2) inhibit
Mtb Dxr by mimicking DXP’s polar character and kill many non-
mycobacterial organisms reliant on this enzyme (Fig. 2).7–9 Our
early work in this area showed that lipophilic analogs of 1 and 2
⇑
Corresponding author. Tel.: +1 202 994 8405; fax: +1 202 994 5873.
E-mail address: cdowd@gwu.edu (C.S. Dowd).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.067

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E. R. Jackson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 649–653
Figure 1. Nonmevalonate pathway of isoprenoid biosynthesis. Dxr (IspC) mediates the conversion of DXP to MEP in the second step.
Figure 2. Fosmidomycin (1), FR900098 (2) and the analogs prepared in this work.
Scheme 1. Reagents and conditions: (a) (EtO)2 P(O)NHOBn, NaH, Nal, TBABr, THF reflux, 18 h; (b) HCl, EtOH, reflux, 5 min; (c) AcCl, TEA, CH2Cl2, rt, 18 h or Ac2O, CH2O2,THF,
rt, 2 h; (d) H2, 10% Pd/C MeOH, 18 h; (e) (i) TMSBr, BSTFA, CH2Cl2, 0 °C to rt, 18 h, (ii) H2O, rt, 18 h, (iii) NaOH aq, rt, 18 h.
Scheme
1
shows the synthetic route used to prepare
presence of acetyl chloride and triethylamine to give compound
6b (52%). Hydrogenation was used to remove the benzyl group,
forming 7 (58%) and 8 (38%). Treatment of 8 with bromotrimeth-
ylsilane, water, and sodium hydroxide gave the mono-sodium salt
9 in quantitative yield.
Scheme 2 was used to prepare analogs with four or five meth-
ylene groups between the nitrogen and phosphorous atoms.
Dibromoalkanes 10a and 10b were treated with triethylphosphite
compounds 7–9, all with a carbon chain of 2 methylene groups.
Compound 319 was reacted with N-(diethoxyphosphoryl)-O-ben-
zylhydroxyl-amine20 in the presence of sodium hydride, sodium
iodide and tetrabutylammonium bromide to form 4 (25%). Further
reaction with concentrated hydrochloric acid gave 5 in quantita-
tive yield.21 Compound 5 was then formylated using acetic anhy-
dride and formic acid to give 6a (71%) or acetylated in the

E. R. Jackson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 649–653
651
Scheme 2. Reagents and conditions: (a) P(OEt)3, microwave 20%, 10–15 min; (b) BnONHAc, NaH, Nal, THF, reflux, 18 h; (c) H2 10% Pd/C, MeOH, rt, 18 h;(d) (i) TMSBr, CH2Cl2,
0 °C to rt, 18 h, (ii) H2O, rt, 18 h, (iii) NaOH aq, rt, 18 h.
Scheme 3. Reagents and conditions: (a) NaH, THF, 60 °C, 18 h; (b) BocNHOBn, NaH, THF, rt, 18 h; (c) BocNHOBn, NaH, Nal, THF, rt, 18 h; (d) (i) AcCl, MeOH, CH2Cl2, rt, 30 min,
(ii) AcCl, Na2CO3, CH2Cl2, rt, 3 h, (e) BCl3, CH2Cl2, À50 °C, 2 h; (f) (i) TMSBr BSTFA, CH2Cl2, 0 °C to rt, 18 h, (ii) H2O, rt, 18 h, (iii) NaOH aq, rt, 18 h.
Scheme 4. Reagents and conditions: (a) (i) TMSBr, CH2Cl2, 0 °C to rt, 18 h, (ii) H2O, rt, 18 h for 23a or H2O, NaOH, rt, 18 h for 23b; (b) chloromethylpivalate, 60 °C TEA/DMF/6–
16 h; (c) H2 10% Pd/C, THF, rt, 18 h for 25 or BCl3, CH2Cl2, À70 °C, 10 h for 26.
in a microwave-assisted Michaelis–Arbuzov reaction to form 11a
(61%) and 11b (64%).22 Acetylated O-benzylhydroxylamine23,24
was treated with sodium hydride and compounds 11a and 11b
to form intermediates 12a (79%) and 12b (37%). Compounds 12a
and 12b underwent hydrogenation to form compounds 13 (34%)
and 14 (49%). Deprotection of the ethyl esters gave compounds
15 and 16 in quantitative yield.
Synthesis of unsaturated FR900098 analog 22 is shown in
Scheme 3. Dibromo compound 1725 was treated with sodium hy-
dride to effect elimination, yielding compound 18 (41%). Boc-pro-
tected O-benzylhydroxylamine26 was reacted with sodium
hydride and then compound 18 to form substituted product 19
(84%). Alternately, compound 19 was prepared directly from 17
in one step using a single treatment of NaH and the amine in
41% yield. Removal of the BOC protecting group in situ and subse-
quent acetylation yielded compound 20 (70%).27 To preserve the
double bond, BCl3 was used to remove the benzyl group of 20,
affording compound 21 (52%).28 Deprotection with bromotrimeth-
ylsilane gave a
/b-unsaturated phosphonic acid 22 (quantitative).29
To assist penetration of compounds across the mycobacterial
cell wall10,30, pivaloyl esters were prepared from two phosphonic
acids (Scheme 4). Diethyl protected intermediates 12a and 20 were
treated with bromotrimethylsilane yielding compounds 23a (87%)
and 23b31 (quantitative). Subsequent reaction with chloromethyl-
pivalate gave esters compounds 24a (6%) and 24b32 (40%). Cata-
lytic hydrogenation removed the benzyl group in saturated
analog 24a, yielding compound 25 (85%). Treatment with BCl3
deprotected unsaturated analog 24b to yield compound 26 (13%).33
Table 1
Effect of chain length on Mtb Dxr inhibition and Mtb MIC
Compound
R
n
Mtb Dxr IC50
(
lM) (% inh at 100
lM)
MIC (lg/mL) 7H9 (GAST)
Fosmidomycin (1)
FR900098 (2)
9
15
16
H
3
3
2
4
5
0.44
2.39
(74%)
(80%)
(86%)
>500
>500
>200 (150)
>200 (>200)
>200 (>200)
CH3
CH3
CH3
CH3
Mtb = Mycobacterium tuberculosis; IC50 = inhibitory concentration at 50%; inh = inhibition; MIC = minimum inhibitory concentration; 7H9 = rich media; GAST = minimal
media.

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E. R. Jackson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 649–653
Table 2
ester 26 show higher potency than the parent compound
Effect of esterification on Mtb MIC
FR900098 (2) on Mtb Dxr inhibition and antitubercular activity.
These data improve our understanding of the Mtb Dxr active site
and its tolerance to length variation between the phosphonate
and retrohydroxamate groups. These results are significant for aid-
ing the rational design of Mtb Dxr inhibitors using the phospho-
nate/retrohydroxamate scaffold and guide the development of
Dxr inhibitors as antitubercular agents.
Compound
R
R1
n
MIC (lg/mL) 7H9 (GAST)
27
7
8
28
29
13
25
14
H
H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2OCOtBu
CH2CH3
CH2OCOtBu
CH2CH3
3
2
2
3
3
4
4
5
400
>500
>500
200–400
50–100
>200 (75)
P200 (150)
>200 (200)
Acknowledgments
CH3
CH3
CH3
CH3
CH3
CH3
This work was supported by the Intramural Research Program
of NIAID/NIH, the George Washington University Department of
Chemistry, the GWU University Facilitating Fund, and NIH
(AI086453 to C.S.D.). R.D.C. was supported by George Mason Uni-
versity’s Department of Chemistry and Biochemistry and the U.S.
Army
Medical
Research
and
Materiel
Command
Table 3
Effect of unsaturation on Mtb Dxr inhibition and Mtb MIC
W23RYX1291N601.
References and notes
1. Dye, C.; Glaziou, P.; Floyd, K.; Raviglione, M. Annu. Rev. Public Health 2013, 34,
271.
2. WHO, ‘Global Tuberculosis Report’, 2012.
3. Rohmer, M. Lipids 2008, 43, 1095.
Compound
R
Mtb Dxr IC50
(lM)
MIC (lg/mL) 7H9 (GAST)
4. Perez-Gil, J.; Rodriguez-Concepcion, M. Biochem. J. 2013, 452, 19.
5. Grawert, T.; Groll, M.; Rohdich, F.; Bacher, A.; Eisenreich, W. Cell. Mol. Life Sci.
2011, 68, 3797.
6. Jackson, E. R.; Dowd, C. S. Curr. Top. Med. Chem. 2012, 12, 706.
7. Mine, Y.; Kamimura, T.; Nonoyama, S.; Nishida, M.; Goto, S.; Kuwahara, S. J.
Antibiot. 1980, 33, 36.
22
21
26
H/Na
CH2CH3
CH2OCOtBu
1.07
NDa
ND
>200 (150)
>200 (150)
9.4 (12.5)
a
ND = not determined.
8. Kuroda, Y.; Okuhara, M.; Goto, T.; Okamoto, M.; Terano, H.; Kohsaka, M.; Aoki,
H.; Imanaka, H. J. Antibiot. 1980, 33, 29.
9. Iguchi, E.; Okuhara, M.; Kohsaka, M.; Aoki, H.; Imanaka, H. J. Antibiot. 1980, 33,
19.
10. Uh, E.; Jackson, E. R.; San Jose, G.; Maddox, M.; Lee, R. E.; Boshoff, H. I.; Dowd, C.
S. Bioorg. Med. Chem. Lett. 2011, 21, 6973.
The analogs were evaluated for inhibition of Mtb Dxr and
growth of Mtb (Tables 1–3). All of the saturated compounds, with
chain lengths between two and five methylene groups, inhibited
Mtb Dxr to some extent (Table 1). Among these acids, compounds
with three methylene groups separating the nitrogen and phos-
phorus atoms (that is, compounds 1 and 2) were the most active.
Not surprisingly, these compounds did not inhibit mycobacterial
11. San Jose, G.; Jackson, E. R.; Uh, E.; Johny, C.; Haymond, A.; Lundberg, L.;
Pinkham, C.; Kehn-Hall, K.; Boshoff, H. I.; Couch, R. D.; Dowd, C. S.
MedChemComm 2013, 4, 1099.
12. McKenney, E. S.; Sargent, M.; Khan, H.; Uh, E.; Jackson, E. R.; San Jose, G.; Couch,
R. D.; Dowd, C. S.; van Hoek, M. L. PLoS One 2012, 7, e38167.
13. Andaloussi, M.; Henriksson, L. M.; Wieckowska, A.; Lindh, M.; Bjorkelid, C.;
Larsson, A. M.; Suresh, S.; Iyer, H.; Srinivasa, B. R.; Bergfors, T.; Unge, T.;
Mowbray, S. L.; Larhed, M.; Jones, T. A.; Karlen, A. J. Med. Chem. 2011, 54, 4964.
14. Deng, L.; Diao, J.; Chen, P.; Pujari, V.; Yao, Y.; Cheng, G.; Crick, D. C.; Prasad, B.
V.; Song, Y. J. Med. Chem. 2011, 54, 4721.
15. Jansson, A. M.; Wieckowska, A.; Bjorkelid, C.; Yahiaoui, S.; Sooriyaarachchi, S.;
Lindh, M.; Bergfors, T.; Dharavath, S.; Desroses, M.; Suresh, S.; Andaloussi, M.;
Nikhil, R.; Sreevalli, S.; Srinivasa, B. R.; Larhed, M.; Jones, T. A.; Karlen, A.;
Mowbray, S. L. J. Med. Chem. 2013, 56, 6190.
16. Verbrugghen, T.; Vandurm, P.; Pouyez, J.; Maes, L.; Wouters, J.; Van Calenbergh,
S. J. Med. Chem. 2013, 56, 376.
17. Hashimoto, M.; Hemmi, K.; Takeno, H.; Kamiya, T. Tetrahedron Lett. 1980, 21,
99.
18. Hemmi, K.; Takeno, H.; Hashimoto, M.; Kamiya, T. Chem. Pharm. Bull. 1982, 30,
111.
19. Balczewski, P.; Pietrzykowski, W. M. Tetrahedron 1996, 52, 13681.
20. Zwierzak, A.; Osowska, K. Synthesis 1984, 3, 223.
21. Blazewska, K.; Gajda, T. Tetrahedron 2003, 59, 10249.
22. Villemin, D.; Simeon, F.; Decreus, H.; Jaffres, P.-A. Phosphorus, Sulfur Silicon
Relat. Elem. 1998, 133, 209.
growth in nutrient-rich media (>200
had a very slight effect when minimal media was used (150
l
g/mL in 7H9), although 9
g/
l
mL in GAST). The polarity of these compounds diminishes penetra-
tion of the lipophilic mycobacterial cell wall.10,30
Diethyl and dipivaloyl esterification of these compounds im-
proved antimycobacterial activity (Table 2). As previously shown,
diethyl esters of 1 and 2 (27 and 28, respectively) are weakly po-
tent inhibitors of Mtb growth with MIC values of 200–400
mL.10 Pivaloyl ester 29 showed improved potency with an MIC of
50–100 g/mL, and this compound was the most potent in the sat-
lg/
l
urated series. Taken together, these data show that linker chains of
two, four or five methylene units are not advantageous for Mtb Dxr
inhibition or inhibition of Mtb cell growth.
The compounds listed in Table 3 were synthesized to examine
the effect of unsaturation on Mtb Dxr inhibition and cell growth.
23. Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck, E.; Jomaa, H.;
Schlitzer, M. Arch. Pharm. 2005, 338, 305.
24. Reichenberg, A.; Wiesner, J.; Weidemeyer, C.; Dreiseidler, E.; Sanderbrand, S.;
Altincicek, B.; Beck, E.; Schlitzer, M.; Jomaa, H. Bioorg. Med. Chem. Lett. 2001, 11,
833.
25. Laureyn, I.; Stevens, C. V.; Soroka, M.; Malysa, P. Arkivoc 2003, iv, 102.
26. Kadi, N.; Oves-Costales, D.; Barona-Gomez, F.; Challis, G. L. Nat. Chem. Biol.
2007, 3, 652.
Interestingly,
a/b-unsaturated compound 22 is a potent inhibitor
of Mtb Dxr with an IC50 of 1.07
lM. Indeed, 22 is more active than
parent compound 2. While 21 and 22 do not inhibit Mtb, the more
lipophilic pivaloyl ester of 22 (compound 26) is a potent inhibitor
of mycobacterial growth with an MIC of 9.4
lg/mL in rich media
and 12.5 g/mL in minimal media. To our knowledge, compound
l
27. Compound 20. Acetyl chloride (1.8 mL, 25.0 mmol) and dry MeOH (0.5 mL)
were added dropwise to 19 (0.19 g, 2.5 mmol) in dry CH2Cl2 (6 mL) under N2.
The reaction mixture was allowed to stir at rt for 30 min. Dry Na2CO3 (0.5 g,
5.0 mmol) and additional acetyl chloride (0.7 mL, 9.8 mmol) were added, and
the reaction mixture was allowed to stir at rt for 3 h. The reaction mixture was
filtered over celite, and the solvent was removed under reduced pressure. The
crude oil was purified by column chromatography using silica gel (CH2Cl2/
MeOH, 49:1) to yield 20 (0.13 g, 0.36 mmol, 77%) as a clear, colorless oil. 1H
NMR (CDCl3, 200 MHz), d (ppm): 7.37 (s, 5H), 6.82–6.57 (m, 1H), 5.90–5.72 (m,
26 displays the most potent antitubercular activity of all com-
pounds that work through a Dxr-mediated mechanism.
Overall, the results collectively indicate that a carbon propyl or
propenyl chain between the nitrogen and phosphorus atoms of
fosmidomycin/FR900098 analogs yields the highest potency. Lipo-
philic esters of these compounds improve their antitubercular
activity. a/b-Unsaturated compound 22 and its lipophilic pivaloyl

E. R. Jackson et al. / Bioorg. Med. Chem. Lett. 24 (2014) 649–653
653
1H), 4.83 (s, 2H), 4.34 (br s, 2H), 4.13–3.99 (m, 4H), 2.13 (s, 3H), 1.34–1.27 (m,
6H). LCMS (ESI) m/z: 705.1 (2 M+Na).
under N2 at 0 °C. The reaction mixture was allowed to warm to room
temperature. After 3.5 h, the mixture was evaporated to dryness, dissolved in
dry CH2Cl2, and evaporated to dryness again (3Â). The resulting residue was
stirred overnight in water (3 mL) and NaOH (5.5 mL, 3 mmol, aq). After 20 h,
the aqueous mixture was washed with CH2Cl2. The organic portion was
separated, and the water was removed by lyophilization to give 23b (0.53 g,
quant.) as white crystals. 1H NMR (CDCl3, 400 MHz), d (ppm): 2.28 (s, 3H), 4.53
(s, 2H), 5.17 (s, 2H), 6.07–6.15 (m, 1H), 6.30–6.40 (m, 1H), 7.65–7.68 (m, 5H).
LCMS (ESI) m/z: 286 (Macid+H), 571 (2Macid+H), 856 (3Macid+H).
28. Compound 21. A solution of 20 (0.117 g, 0.34 mmol) in dry CH2Cl2 (6.0 mL) was
cooled to À50 °C and BCl3 (1.4 mL, 1 M in CH2Cl2) was added dropwise, and the
reaction mixture was allowed to stir for 2 h. The reaction was quenched with
saturated NaHCO3 (aq, 9.0 mL) and allowed to warm to rt. The aqueous
solution was extracted with CH2Cl2. The organic fractions were combined,
dried over MgSO4, filtered and the solvent was removed under reduced
pressure. The resulting crude residue was purified using an Isolera Flash
Chromatography system and a silica column (EtOAc/MeOH, 49:1) to yield 21
(45 mg, 0.18 mmol, 52%) as a light yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm): 1.28 (t, 6H), 2.15 (s, 3H), 4.02 (q, 4H),4.43–4.25 (m, 2H), 5.84 (t,
J = 18.8 Hz, 1H), 6.83–6.52 (m, 1H), 9.88 (br s, 1H). 13C NMR (50 MHz, CDCl3) d
(ppm): 16.30, 20.36, 50.47 (d, J = 27.2 Hz), 62.37 (d, J = 7.1 Hz), 120.02, 147.62,
172.66. LCMS (ESI) m/z 252.1 (M+H).
32. Compound 24b. Chloromethylpivalate (2.15 mL, 15 mmol) was added to
a
stirred solution of 23b (0.49 g, 1.5 mmol) and triethylamine (0.45 mL, 3 mmol)
in DMF (40 mL). The reaction mixture was heated to 60 °C for 16 h. Water
(50 mL) was added, and the aqueous layer was extracted with CH2Cl2. The
organic layer was washed with brine, dried over MgSO4, and the solvent was
removed under reduced pressure. The crude oil was purified by column
chromatography using silica gel and CH2Cl2/EtOAc to yield 24b (0.22 g, 28%).
1H NMR (CDCl3, 400 MHz), d (ppm): 1.20 (s, 18H), 2.12 (s, 3H), 4.33 (br s, 2H),
4.82 (s, 2H), 5.65 (d, J = 12.8 Hz, 4H), 5.80–5.89 (m, 1H), 6.70–6.81 (m, 1H),
7.35–7.38 (m, 5H). LCMS (ESI) m/z: 536 (M+Na).
29. Compound 22. N,O-Bis(trimethylsilyl)trifluoroacetamide (0.18 mL, 0.67 mmol)
was added to 21 (0.03 g, 0.12 mmol) in CH2Cl2 (0.60 mL) under N2. The reaction
mixture was allowed to stir at rt for 20 min. The reaction mixture was cooled
to 0 °C, and bromotrimethylsilane (0.18 mL, 1.34 mmol) was added dropwise.
The reaction was allowed to warm to rt and was stirred overnight under N2.
Ethyl bromide and excess silylating agent were removed under reduced
pressure, and the residue was dissolved in aqueous NaOH (0.68 mL, 7.8 mg/
mL) and stirred overnight. The mixture was extracted between H2O and
CH2Cl2. The aqueous portions were combined, and the solvent was removed by
lyophilization to give 22 (0.03 g, 0.12 mmol, quant.) as a yellow solid. 1H NMR
(400 MHz, D2O) d (ppm): 2.36 (s, 3H), 4.61–4.46 (m, 2H), 6.19–6.06 (m, 1H),
33. Compound 26. BCl3 (1 M in CH2Cl2, 0.88 mL) was added dropwise to a stirred
solution of 24b (190 mg, 0.37 mmol) in dry CH2Cl2 (5 mL) under N2 at À78 °C.
After 10 h, the reaction mixture was poured into satd. NaHCO3 (aq) and was
extracted with CH2Cl2. The organic layers were combined and washed with
brine, dried over MgSO4, and the solvent was removed under reduced pressure.
The crude oil was purified by column chromatography using EtOAc, CH2Cl2,
and MeOH. The oil was further purified over a silica plug, washed with hexanes
and CH2Cl2, and then eluted with EtOAc to give 26 as a pale yellow oil (21 mg,
13.4%). 1H NMR (CDCl3, 400 MHz), d (ppm): 1.22 (s, 18H), 2.19 (s, 3H), 4.41 (s,
6.56–6.43 (m, 1H). 13C NMR (101 MHz, D2O)
d (ppm): 19.34, 50.81 (d,
J = 23.7 Hz), 126.91, 137.79, 174.18. HRMS (ESI) m/z calcd for C10H18N2NaO10P2
(2 M+Na]): 411.0328, found: 411.0334.
2H), 5.61–5.69 (m, 4H), 5.87–5.97 (m, 1H), 6.71–6.84 (m, 1H), 8.61 (s, 1H). 13
C
30. Brown, A. C.; Parish, T. BMC Microbiol. 2008, 8, 78.
31. Compound 23b. Trimethylsilylbromide (1.75 mL, 11.7 mmol) was added
dropwise to a stirring solution of 20 (0.5 g, 1.5 mmol) in dry CH2Cl2 (20 mL)
NMR (CDCl3, 100 MHz), d (ppm): 20.47, 26.93, 38.88, 50.30 (d, J = 26.5 Hz),
81.70 (d, J = 5.3 Hz), 117.95 (d, J = 188.2 Hz), 148.50, 172.87, 177.30. LCMS (ESI)
m/z: 446 (M+Na), 847 (2 M+H), 869 (2 M+Na).